Synthesis and binding affinities of methylvesamicol analogs for the acetylcholine transporter and sigma receptor

Article abstract of DOI:10.1016/j.bmc.2005.11.044

We synthesized methylvesamicol analogs 13-16 and investigated the binding characteristics of 2-[4-phenylpiperidino]cyclohexanol (vesamicol) and methylvesamicol analogs 13-16, with a methyl group introduced into the 4-phenylpiperidine moiety, to sigma receptors (σ-1, σ-2) and to vesicular acetylcholine transporters (VAChT) in membranes of the rat brain and liver. In competitive inhibition studies, (-)-o-methylvesamicol [(-)-OMV] (13) (K_i = 6.7 nM), as well as (-)-vesamicol (K_i = 4.4 nM), had a high affinity for VAChT. (+)-p-Methylvesamicol [(+)-PMV] (16) (K_i = 3.0 nM), as well as SA4503 (K_i = 4.4 nM), reported as a σ-1 mapping agent for positron emission tomography (PET), had a high affinity for the σ-1 receptor. The binding affinity of (+)-PMV (16) for the σ-1 receptor (K_i = 3.0 nM) was about 13 times higher than that for the sigma-2 (σ-2) receptor (K_i = 40.7 nM). (+)-PMV (16) (K _i = 199 nM) had a much lower affinity for VAChT than SA4503 (K _i = 50.2 nM) and haloperidol (K_i = 41.4 nM). These results showed that the binding characteristics of (-)-OMV (13) to VAChT were similar to those of (-)-vesamicol and that (+)-PMV (16) bound to the σ-1 receptor with high affinity. In conclusion, (-)-OMV (13) and (+)-PMV (16), which had a suitable structure, with a methyl group for labeling with ¹¹C, may become not only a new VAChT ligand and a new type of σ receptor ligand, respectively, but may also become a new target compound of VAChT and the σ-1 receptor radioligand for PET, respectively.

Full text of DOI:10.1016/j.bmc.2005.11.044

Bioorganic & Medicinal Chemistry 14 (2006) 2620–2626
Synthesis and binding affinities of methylvesamicol analogs
for the acetylcholine transporter and sigma receptor
Kazuhiro Shiba,a,* Kazuma Ogawa,a Kiichi Ishiwata,b Kazuyoshi Yajimac
and Hirofumi Moria
aDivision of Tracer Kinetics, Advanced Science Research Center, Kanazawa University, Kanazawa, Japan
bPositron Medical Center, Tokyo Metropolitan Institute of Gerontology, Tokyo, Japan
cMedical and Pharmacological Research Center Foundation, Hakui, Japan
Received 3 August 2005; revised 22 November 2005; accepted 22 November 2005
Available online 24 January 2006
Abstract—We synthesized methylvesamicol analogs 13–16 and investigated the binding characteristics of 2-[4-phenylpiperidi-
no]cyclohexanol (vesamicol) and methylvesamicol analogs 13–16, with a methyl group introduced into the 4-phenylpiperidine moi-
ety, to sigma receptors (r-1, r-2) and to vesicular acetylcholine transporters (VAChT) in membranes of the rat brain and liver. In
competitive inhibition studies, (ꢀ)-o-methylvesamicol [(ꢀ)-OMV] (13) (Ki = 6.7 nM), as well as (ꢀ)-vesamicol (Ki = 4.4 nM), had a
high affinity for VAChT. (+)-p-Methylvesamicol [(+)-PMV] (16) (Ki = 3.0 nM), as well as SA4503 (Ki = 4.4 nM), reported as a r-1
mapping agent for positron emission tomography (PET), had a high affinity for the r-1 receptor. The binding affinity of (+)-PMV
(16) for the r-1 receptor (Ki = 3.0 nM) was about 13 times higher than that for the sigma-2 (r-2) receptor (Ki = 40.7 nM). (+)-PMV
(16) (Ki = 199 nM) had a much lower affinity for VAChT than SA4503 (Ki = 50.2 nM) and haloperidol (Ki = 41.4 nM). These results
showed that the binding characteristics of (ꢀ)-OMV (13) to VAChT were similar to those of (ꢀ)-vesamicol and that (+)-PMV (16)
bound to the r-1 receptor with high affinity. In conclusion, (ꢀ)-OMV (13) and (+)-PMV (16), which had a suitable structure, with a
methyl group for labeling with 11C, may become not only a new VAChT ligand and a new type of r receptor ligand, respectively, but
may also become a new target compound of VAChT and the r-1 receptor radioligand for PET, respectively.
ꢀ 2005 Elsevier Ltd. All rights reserved.
1. Introduction
ity for VAChT or sigma receptors.6 As a result, we
found that radioiodinated (+)-p-iodovesamicol [(+)-p-
IV] radioiodinated at the para-position into the 4-phen-
ylpiperidine moiety of the (+)-enantiomer of vesamicol
bound to sigma receptor (r-1) with high affinity and
high selectivity.9 In this study, with the aim of extending
human VAChT or sigma receptor imaging studies to a
higher level of precision with positron emission tomog-
raphy (PET), we synthesized methylvesamicol analogs
that had a suitable structure for labeling with 11C and
investigated the in vitro binding characteristics of these
analogs to VAChT and sigma receptors (r-1, r-2). Fur-
thermore, we synthesized trialkylstannyl vesamicol ana-
logs, which were used as precursors for the synthesis of
11CH3-labeled vesamicol analogs.
Several radiolabeled analogs of vesamicol1–5 have been
proposed as VAChT mapping agents for diagnosing
dementia characterized by the degeneration of the cho-
linergic neurotransmitter system, such as Alzheimer’s
disease. We also reported that radioiodinated
(ꢀ)-o-iodovesamicol [(ꢀ)-o-IV], radioiodinated at the
ortho-position of the 4-phenylpiperidine moiety of (ꢀ)-
enantiomer of vesamicol, was suitable as a VAChT
mapping agent for SPECT.6,7 On the other hand,
Efange et al.2 and Custer et al.8 reported that there were
several vesamicol analogs showing great affinity not
only for VAChT, but also for sigma receptors. Then,
we investigated the relationships between the position
of the substituent into the 4-phenylpiperidine moiety
of vesamicol and a kind of optical isomer, and the affin-
2. Results
2.1. Chemistry
Keywords: Vesamicol analogs; Sigma receptor; Acetylcholine
transporter.
*
The key intermediates for the synthesis of (ꢀ)-o-meth-
ylvesamicol ((ꢀ)-OMV) (13) and (+)-p-methylvesamicol
Corresponding author. Tel.: +81 76 265 2474; fax: +81 76 234
4245; e-mail: shiba@med.kanazawa-u.ac.jp
0968-0896/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2005.11.044

K. Shiba et al. / Bioorg. Med. Chem. 14 (2006) 2620–2626
2621
((+)-PMV) (16) were 4-(2-methylphenyl)piperidine (9)
and 4-(4-methylphenyl)piperidine (10), respectively.
These methylvesamicol analogs were prepared as out-
lined in Scheme 1. 2-Methylbenzaldehyde (3) was pre-
pared from 2-bromobenzaldehyde (1) by a three-step
reaction, the protection of the aldehyde with ethylene
glycol, formation of the Grignard reagent, and methyl-
ation with methyl iodide. The reaction of 2-methyl-
benzaldehyde (3) with ethyl cyanoacetate under
Claizen–Schmidt conditions gave 5 in 55.3% yield by
purification using column chromatography on silica
gel. The conjugate addition of the diethylmalonate
carbanion onto 5, followed by acid-catalyzed hydroly-
sis, provided the crude diacid. The diacid was convert-
ed first to the corresponding diester and then to imide
7 with an overall yield of 61.2%. Reduction of the
imide 7 with borane-tetrahydrofuran (BH3–THF) affor-
ded the desired key intermediate, 9, in 66.5% yield. The
racemic o-methylvesamicol (11) was obtained in the
usual manner by refluxing 9 with cyclohexene oxide
in ethanol. Each enantiomer of o-methylvesamicol (13
and 14) was obtained by recrystallizing the correspond-
ing diastereoisomeric salts with (ꢀ)-di-p-toluoyl-L-tar-
taric acid and (+)-di-p-toluoyl-D-tartaric acid,
respectively. Each enantiomer of p-methylvesamicol, 15
and 16, was prepared by the same method as for 13
and 14 with 4-bromobenzaldehyde (2). Each enantiomer
of the methylvesamicol analogs 13–16 from the corre-
sponding starting materials (1 and 2) via 10-step reac-
tions produced respective overall yields of 1.3–4.1%.
Trialkylstannyl compounds, 19 and 20, used as precur-
sors for 11CH3-labeled tracers, were prepared by a palla-
dium-catalyzed reaction of the corresponding
iodovesamicols, 17 and 18 (Scheme 2). Modest yields
of trialkylstannyl compounds (33–71%) were obtained
after purification through thin-layer chromatography
on silica gel.
2.2. Binding studies
The binding affinities of methylvesamicol analogs and
reference compounds to the VAChT binding sites and
sigma receptors (r-1, r-2) are shown in Table 1.
o-Methylvesamicol, as well as vesamicol, bound enanti-
oselectively to the VAChT binding sites. The binding
R
1
R
1
R
1
COOEt
CN
a
b
c
d
R
CHO
R
CHO
2
2
R
2
3
4
R1 = CH3, R2 = H
R1 = H, R2 = CH3
1
2
R1 = Br, R2 = H
R1 = H, R2 = Br
5
6
R1 = CH3, R2 = H
R1 = H, R2 = CH3
e
R
R
1
1
O
R
1
COOH
COOH
g
f
h
R
NH
R
2
NH
2
R
2
9
R1 = CH3,
R2 = H
O
7
8
R1 = CH3, R2 = H
R1 = H, R2 = CH3
i
10 R1 = H,
R2 = CH3
R
R
1
1
HO
N
13 R1 = CH3, R2 = H
14 R1 = H, R2 = CH3
(-)
R
R
HO
N
2
j
1
(+/-)
R
2
HO
N
k
15 R1 = CH3, R2 = H
16 R1 = H, R2 = CH3
11 R1 = CH3, R2 = H
12 R1 = H, R2 = CH3
(+)
R
2
Scheme 1. Synthesis of methylvesamicol analogs. Reagents and conditions: (a) ethylene glycol, p-TsOH, benzene, reflux; (b) Mg, THF, CH3I, reflux;
(c) 5% HCl, rt; (d) ethylcyanoacetate, AcOH, pyrrolidine, reflux; (e) i—NaH, diethylmalonate, THF, 4 ꢁC, ii—6 N HCl, reflux; (f) SOCl2, MeOH,
4 ꢁC; (g) urea, NaOEt, EtOH, reflux; (h) BH3–THF, reflux; (i) cyclohexene oxide, EtOH, reflux; (j) (ꢀ)-di-p-toluoyl-L-tartaric acid, acetone, rt; (k)
(+)-di-p-toluoyl-D-tartaric acid, acetone, rt.
R1
R1
HO
N
HO
N
m
R2
R2
17 R1 = I, R2 = H
18 R1 = H, R2 = I
19 R1 = Sn(CH3)3, R2 = H
20 R1 = H, R2 = Sn(n-Bu)3
Scheme 2. Synthesis of trialkylstannyl-vesamicol analogs. Reagents: (m) [(alkyl)3Sn]2, (Ph3P)4Pd(0), toluene, reflux.

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K. Shiba et al. / Bioorg. Med. Chem. 14 (2006) 2620–2626
Table 1. Affinities of vesamicol derivatives and sigma receptor ligands
for vesicular acetylcholine transporter (VAChT) and sigma receptors
(r-1, r-2)
binding for VAChT. The affinity for VAChT was in
the order of (ꢀ)-vesamicol = (ꢀ)-OMV (13) >
(+)-OMV
(14) > (ꢀ)-PMV
(15) > haloperidol >
VAChT (Ki)
Sigma receptor
SA4503 > (+)-vesamicol > DTG > (+)-PMV (16) > (+)-
pentazocine. The VAChT binding affinity of (ꢀ)-OMV
(13) (Ki = 6.7 nM) was slightly less than that of
(ꢀ)-vesamicol (Ki = 4.4 nM). However, the affinity of
(ꢀ)-OMV (13) to VAChT was thought to be sufficiently
high for the VAChT imaging.
r-1 (Ki)
r-2 (Ki)
(ꢀ)-Vesamicol
(+)-Vesamicol
(ꢀ)-OMV
(+)-OMV
4.4 0.6
135 14
73.8 11.2
31.5 11.3
33.7 5.9
10.7 0.2
8.1 2.0
3.0 0.2
4.4 1.0
2.6 0.8
5.5 2.0
—
346 37
330 36
266 28
218 20
42.4 6.3
40.7 2.9
242 17
167 19
2470 150
20.7 4.8
6.7 0.4
22.5 2.0
22.9 0.4
199 32
(ꢀ)-PMV
Then, the binding affinity of (ꢀ)-OMV (13) to the
VAChT binding sites (Ki = 6.7 nM) was much higher
than those of the r-1 receptor (Ki = 33.7 nM) and the
r-2 receptor (Ki = 266 nM), which showed that (ꢀ)-
OMV bound to VAChT binding sites with high specific-
ity. Thus, (ꢀ)-OMV (13), which had a suitable structure
with a methyl group for labeling with 11C, had similar
characteristics for VAChT as (ꢀ)-vesamicol. Because
the labeling of vesamicol with 11C is structurally diffi-
cult, (ꢀ)-OMV (13) may become a new target com-
pound of the VAChT radioligand for PET.
(+)-PMV
SA4503
50.2 7.2
41.4 17.6
315 121
165 26
Haloperidol
(+)-Pentazocine
DTG
Ki values derived from IC50 values according to the equation:
Ki = IC50/(1 + C/Kd), where C is the concentration of the radioligand
and each Kd is the dissociation constant of the corresponding radio-
ligand (Kd of (ꢀ)-[3H]vesamicol to VAChT (Kd = 7.40 nM), Kd of
[3H]pentazocine to r-1 (Kd = 19.9 nM), Kd of [3H]DTG to
r-2 (Kd = 22.3 nM)).
Regarding the introduction of the methyl group into the
para-position of the 4-phenylpiperidine moiety, p-meth-
ylvesamicol (15 and 16) exhibited a higher affinity for
sigma receptors (r-1, r-2) and a lower affinity for
VAChT binding sites than vesamicol and o-methylve-
samicol (3 and 14). Namely, the introduction of the
methyl group into the para-position of vesamicol caused
its binding affinity for sigma receptors (r-1, r-2) to in-
crease, and its binding affinity for VAChT binding sites
to decrease. The p-methylvesamicol bound enantioselec-
tively to the r-1 receptor. The r-1 receptor binding
affinity of (+)-PMV (16) (Ki = 3.0 nM) was greater than
that of (ꢀ)-PMV (15) (Ki = 8.1 nM). Furthermore, the
r-1 receptor binding of (+)-PMV (16) was comparable
to that of haloperidol (Ki = 2.6 nM), and thought to
be sufficiently high to be used as a r-1 receptor imaging
agent, because the selectivity of (+)-PMV (16) (r-2/
r-1 = about 14) for the r-2 and r-1 receptors was com-
parable to that of (+)-p-IV (r-2/r-1 = about 16), which
was reported previously to be a sigma-1 receptor li-
gand.9 (+)-PMV (16) (Ki = 199 nM) showed a lower
affinity for VAChT binding sites than SA4503
(Ki = 50.2 nM) and haloperidol (Ki = 41.4 nM). (+)-Pen-
tazocine and haloperidol are thought to bind to other
neuroreceptors.15,16 In addition, (+)-[3H]pentazocine
showed low accumulation in the rat brain.17 (+)-PMV
(16) has a structure similar to that of (+)-p-IV. Judging
from the above, (+)-PMV (16), as well as SA4503, was
affinity of (ꢀ)-OMV (13) (Ki = 6.7 nM) to the VAChT
binding sites was greater than that of (+)-OMV (14)
(Ki = 22.5 nM). Also, the binding affinity of (ꢀ)-OMV
(13) (Ki = 33.7 nM) to the r-1 receptor was less than
that of (+)-OMV (14) (Ki = 10.7 nM). (+)-PMV (16)
(Ki = 3.0 nM) displayed a slightly higher affinity for
the r-1 receptor than SA4503 (Ki = 4.4 nM) and
(+)-pentazocine (Ki = 5.5 nM), which are known as sig-
ma ligands. The binding affinity of (+)-PMV (16) to the
r-1 receptor (Ki = 3.0 nM), was about 14 times higher
than the sigma-2 (r-2) receptor (Ki = 40.7 nM). Also,
(+)-PMV (16) displayed a lower affinity for the VAChT
binding sites (Ki = 199 nM) than SA4503 (Ki = 50.2 nM)
and haloperidol (Ki = 41.4 nM).
3. Discussion
Radiolabeled vesamicol analogs have been proposed not
only to be VAChT, but also sigma receptor, mapping
agents.10–13 In this study, we synthesized methylvesami-
col analogs (13–16) and investigated the binding affinity
of these analogs to VAChT binding sites or sigma recep-
tors (r-1, r-2). First, we attempted to synthesize
4-(2-methylphenyl)piperidine (9) and 4-(2-methylphe-
nyl)piperidine (10) as the key intermediates for the syn-
thesis of methylvesamicol analogs (13–16). To introduce
a methyl group into the ortho- or para-position of the 4-
phenylpiperidine, two methylbenzaldehydes (3 and 4)
were prepared from bromobenzaldehydes (1 and 2) with
a Grignard reaction, and 9 and 10 were prepared from
the corresponding compounds (3 and 4) via a five-step
reaction. Methylvesamicol analogs (13–16) were synthe-
sized from these key intermediates (9 and 10) in the same
manner as reported by Roger et al.14
thought to be
a superior r-1 receptor ligand.
[11C]SA4503 has already been reported to be a potential
radioligand for mapping of the r-1 receptor.18,19 In the
future, (+)-[11C]PMV should be evaluated as to its po-
tential as a PET radioligand for mapping the r-1 recep-
tor, as compared with [11C]SA4503.
Trialkylstannyl compounds (19 and 20), precursors of
(ꢀ)-[11C]OMV and (+)-[11C]PMV, were prepared from
the corresponding iodovesamicol reported previously.6,9
(ꢀ)-[11C]OMV and (+)-[11C]PMV, labeled with 11C, will
be synthesized by methylation of the corresponding tri-
alkylstannyl compounds (19 and 20) with [11C]methyl
The results of binding studies showed that the introduc-
tion of a methyl group, as well as iodine, into the ortho-
position of the 4-phenylpiperidine moiety had no
influence on the affinity of methylvesamicol analog

K. Shiba et al. / Bioorg. Med. Chem. 14 (2006) 2620–2626
2623
iodide in
reaction.20
a
palladium-promoted cross-coupling
mixture was cooled in an ice-bath and treated with
25% aq NH4Cl (150 mL) and extracted with ether. The
organic extract was dried over anhydrous Mg2SO4 and
concentrated under reduced pressure to provide the cor-
responding methyl-ketal as a yellow syrup. This syrup
was dissolved in acetone (50 mL) and treated with 5%
HCl. The mixture was stirred for 30 min at room tem-
perature. After removing the acetone under reduced
pressure, the reaction mixture was diluted with water,
and then made alkaline with K2CO3. The mixture was
extracted with ethyl acetate. The organic solvent was
dried over MgSO4 and evaporated to leave a syrup,
which was purified by column chromatography on silica
gel using ethyl acetate/hexane (1:9, v/v) as the eluent to
yield 3 (11.5 g, 70.8% from 2-bromobenzaldehyde, 1).
In conclusion, the introduction of the methyl group, as
well as iodine, into the ortho- or para-position of vesam-
icol varied the binding characteristics of methylvesami-
col analogs for VAChT binding sites and sigma
receptors (r-1, r-2). (ꢀ)-OMV (13) and (+)-PMV (16)
may become not only a new VAChT ligand and a new
type of r receptor ligand, respectively, but may also be-
come a new target compound of VAChT and a r-1
receptor positron imaging agent, respectively. At pres-
ent, (ꢀ)-[11C]OMV and (+)-[11C]PMV are prepared by
the substitution of the trialkylstannyl group with
[11C]methyl iodide in a palladium-promoted cross-cou-
pling reaction20 and we evaluated these in vivo charac-
teristics in the brain of rats and monkeys.
Compound 3: 1H NMR (CDCl3): d 2.66 (s, 3H), 7.84–7.31
(m, 4H), 10.26 (br s, 1H). Mass spectrum (m/e): 120 [M]+.
4. Experimental
4.2. Ethyl 2-cyano-3-(2-methylphenyl)acrylate (5)
Melting points were measured with a Yanagimoto
micromelting point apparatus. NMR spectra were
recorded on a JEOL JNM-GSX500 (taken in deuterated
chloroform with tetramethylsilane as the internal stan-
dard). Mass spectra(MS) recorded on a JEOL JMS-
SX102 spectrometer coincided well with the proposed
structures. Elemental analyses were performed on a
Yanaco CHN Corder MT-5; all results were within
0.3% of theoretical values. Specific rotation was ob-
tained on a Nippon Bunko DIP-181 digital polarimeter.
(ꢀ)-[3H]Vesamicol (1.30 TBq/mmol), [3H]1,3-di-tolyl-
guanidine ([3H]DTG) (1.1 TBq/mmol), [3H]pentazocine
(1.0 TBq/mmol), and [125I]sodium iodide (644 GBq/mg)
were purchased. Other drugs and reagents, except for
SA4503, were purchased from Sigma-Aldrich Japan
(Tokyo, Japan). SA4503 was prepared by M’s Science
(Kobe, Japan).
Glacial acetic acid (5.6 mL) and pyrrolidine (3.07 mL)
were added to a mixture of 3 (17.8 g, 148 mmol) and eth-
ylcyanoacetate (20.1 g, 177.7 mmol) in benzene
(150 mL). The resulting mixture was refluxed with the
azeotropic removal of water for 120 min, cooled to
room temperature, diluted with benzene (50 mL), and
washed consecutively with water (100 mL) and saturated
NaHCO3 (100 mL). The organic extract was dried over
anhydrous MgSO4 and evaporated under reduced pres-
sure. The crude product was purified by column chro-
matography on silica gel with ethyl acetate/hexane
(1:4, v/v). The solvent was removed under reduced pres-
sure to provide 17.62 g (55.3%) of 5.
Compound 5: 1H NMR (CDCl3): d 1.52–1.29 (t,
J = 7.02 Hz, 3H), 2.45 (s, 3H), 4.58–4.22 (dd,
J = 7.02 Hz, J = 14.3 Hz, 2H), 7.38–7.34 (m, 3H), 8.56
(s, 1H), 8.18–8.09 (m, 1H). Mass spectrum (m/e): 215
(M+).
4.1. 2-Methylbenzaldehyde (3)
A
mixture of 2-bromobenzaldehyde (1) (25 g,
4.3. 3-(2-Methylphenyl)piperidine-2,6-dione (7)
135 mmol), ethylene glycol (41.9 g, 675 mmol), and
p-toluenesulfonic monohydrate (2.57 g, 13.5 mmol) in
benzene (300 mL) was refluxed for 3 h with a Dean–
Stark phase separating head and the water was removed
as formed. The cooled reaction mixture was diluted with
an equal volume of ether, and this mixture was washed
with 10% sodium carbonate solution and then with
water. The washed organic layer was dried by filtration
through anhydrous Mg2SO4 and evaporated to near
dryness under reduced pressure to provide a quantita-
tive yield of the crude corresponding ketal.
Sodium hydride (6.36 g, of a 60% oil immersion,
160 mmol) was washed with dry THF (20 mL) under ar-
gon and resuspended in dry THF (50 mL). A solution of
diethyl malonate (25.92 g, 162 mmol) in dry THF
(60 mL) was added dropwise with cooling (ice bath),
over 20 min. The ice bath was removed and the resulting
solution was stirred for an additional 15 min. At this
time, a solution of 5 (17.12 g, 79.6 mmol) in dry THF
(100 mL) was added dropwise over 30 min. After stir-
ring for 13 h, the reaction mixture was treated with
20% aq NH4Cl (200 mL) and the layers were separated.
The aq layer was reextracted with ethyl acetate and set
aside. The combined organic extracts were dried over
anhydrous MgSO4 and concentrated to a syrup. The lat-
ter was treated with 6 N HCl (200 mL) and the mixture
was refluxed for 60 h. Upon cooling, the diacid crystal-
lized from the solution. Thionyl chloride (21.62 g,
181.7 mmol) was added dropwise over 30 min to a
cooled (ice bath) solution of the crude diacid product
in dry MeOH (90 mL). Cooling was continued for an
Next, in a three-necked flask fitted with a reflux con-
denser and a separatory funnel containing Mg turnings
(3.64 g, 150 mmol) and dry THF (50 mL), the crude dry
ketal (22.9 g, 100 mmol) in dry THF (50 mL) was added
dropwise under Argon gas. After the addition was com-
plete, the whole mixture was refluxed for 1 h. After the
flask was cooled in ice-cold water, methyl iodide (71 g,
500 mmol) in dry THF (60 mL) was added dropwise
and the mixture was refluxed for 2 h. The reaction

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K. Shiba et al. / Bioorg. Med. Chem. 14 (2006) 2620–2626
1
additional 60 min and the ice bath was then removed.
Stirring was then continued for 16 h, at which time the
solvent was removed under reduced pressure and the
resulting syrup was redissolved in CH2Cl2 (20 mL).
The solution was washed with saturated aq NaHCO3
(2· 75 mL), dried over anhydrous MgSO4, and concen-
trated under reduced pressure to provide the crude dies-
ter as a syrup. The latter was used without further
purification.
Compound 11: mp: 116–118 ꢁC. H NMR (CDCl3): d
1.38–1.19 (m, 4H), 1.84–1.71 (m, 8H), 2.33–2.07 (m,
3H), 2.33 (s, 3H), 2.86–2.69 (m, 3H), 3.70–3.42 (m,
1H), 7.25–7.09 (m, 4H). Mass spectrum (m/e): 273
(M+). Anal. Calcd for C18H27NO: C, 79.07; H, 9.95;
N, 5.12. Found: C, 78.88; H, 10.15; N, 5.06.
4.6. (ꢀ)-2-(4-(2-Methylphenyl)piperidino)cyclohexanol
((ꢀ)-OMV) (13)
A mixture of urea (5.28 g, 88.0 mmol) and the diester in
dry EtOH (50 mL) was quickly added to a solution of
sodium ethylate (80 mL) prepared from 2.03 g
(0.088 g-atm) of sodium. The resulting mixture was re-
fluxed for 17 h, cooled to room temperature, and filtered
to remove the insoluble material, which was later shown
to be 7. The latter was washed consecutively with 10%
aq NH4Cl and ethyl acetate to yield 9.90 g (61.2%) of
the imide.
Racemic o-methylvesamicol (11) (1.63 g, 5.96 mmol) in
25 mL of acetone was added dropwise with stirring to
(ꢀ)-di-p-toluoyl-L-tartaric acid monohydrate (2.54 g,
6.56 mmol) in 25 mL of acetone and set aside to crystallize
at 23 ꢁC. Crude crystals were collected by filtering and
recrystallized from EtOH. The yield of product 13 was
0.541 g (66.5%). The filtrate was concentrated under re-
duced pressure and used to obtain the (+)-isomer of 11.
Compound 13: mp: 116–118. Anal. Calcd for
C18H27NO: C, 79.07; H, 9.95; N, 5.12. Found: C,
Compound 7: 1H NMR (CD3OD): d 2.36 (s, 3H), 2.83–
2.70 (m, 4H), 3.76–3.52 (m, 1H), 7.19 (s, 4H). Mass spec-
trum (m/e): 203 (M+).
30
D
78.94; H, 10.20; N, 5.06. Specific rotation: ½aꢁ ꢀ28.4
(c = 0.502, CHCl3).
4.4. 4-(2-Methylphenyl)piperidine (9)
4.7. (+)-2-(4-(2-Methylphenyl)piperidino)cyclohexanol
((+)-OMV) (14)
150 mL (150 mmol) of 1.0 M BH3–THF was introduced
into a freshly dried three-necked flask fitted with a reflux
condenser, magnetic stirrer, and dropping funnel; this
flask was placed in an ice-water bath. Then, the com-
pound 7 (9.46 g, 46.6 mmol) in dry THF (25 mL) was
added to the borane solution under argon. After the
addition was complete, the reaction mixture was stirred
for 20 min at below 20 ꢁC and then refluxed for 19 h. At
this time, the mixture was allowed to cool to room tem-
perature, and the reaction was quenched by the careful
addition of 6 N HCl (20 mL). After the evolution of
hydrogen had subsided, the mixture was concentrated
under reduced pressure and the residue was treated with
more 6 N HCl (100 mL). The resulting solution was re-
fluxed for 22 h, cooled to room temperature, and con-
centrated to a white solid that was redissolved in water
(100 mL). The solution was adjusted to pH 11 with
NaOH pellets and extracted successively with CH2Cl2.
The organic extracts were dried over anhydrous MgSO4
and concentrated under reduced pressure to provide
6.60 g (81.0%) of 9 as a syrup, which solidified upon
standing.
The filtrate containing the (+)-isomer of 11 was added
dropwise with stirring to (+)-di-p-toluoyl-D-tartaric acid
monohydrate (5.54 g, 6.56 mmol) in 25 mL of acetone
and set aside to crystallize at 23 ꢁC. Crystals were col-
lected by filtering and recrystallized from EtOH. The
yield of product 14 was 0.168 g (20.6%).
Compound 14: mp: 116–118 ꢁC. Anal. Calcd for
C18H27NO: C, 79.07; H, 9.95; N, 5.12. Found: C, 79.01;
30
D
H, 10.04; N, 4.97. Specific rotation: ½aꢁ
+28.3
(c = 0.504, CHCl3).
4.8. ( )-2-(4-(4-Methylphenyl)piperidino)cyclohexanol
(( )-p-methylvesamicol) (( )-PMV) (12)
Synthesis was carried out as for compound 12, with
4-bromobenzaldehyde (2). Compound 12 from 2 via
an eight-step reaction produced respective overall yields
of 5.0%.
Compound 12: mp: 128–130 ꢁC. NMR (CDCl3): d 1.23
(4H, m), 1.57–1.84 (8H, m), 2.13 (1H, m), 2.22–2.2
(2H, m), 2.32 (3H, s), 2.43–2.45 (1H, m), 2.72–2.74
(2H, m), 2.92 (1H, m), 3.39 (1H, m), 7.12 (4H, s). Anal.
Calcd for C18H27NO: C, 79.07; H, 9.95; N, 5.12. Found:
C, 78.84; H, 9.96; N, 4.91. Mass spectrum (m/e): 273
(M+).
Compound 9: 1H NMR (CD3OD): d 2.05 (m, 4H), 2.35
(s, 3H), 7.19 (s, 4H), 4.15–3.54 (m, 5H). Mass spectrum
(m/e): 175 (M+).
4.5. ( )-2-(4-(2-Methylphenyl)piperidino)cyclohexanol
(( )-o-methylvesamicol) (( )-OMV) (11)
4.9. (ꢀ)-2-(4-(4-Methylphenyl)piperidino)cyclohexanol
((ꢀ)-p-methylvesamicol) ((ꢀ)-PMV) (15)
Compound 9 (4.65 g, 26.6 mmol) and cyclohexene oxide
(10.1 mL, 100 mmol) were dissolved in 30 mL of dry
EtOH and refluxed for 20 h. Upon cooling of the solu-
tion, white crystals formed, which were collected by fil-
tering and washed with cold EtOH. The crude product
was recrystallized from EtOH. Yield of product 11
was 2.32 g (31.9%).
This compound, 15, was prepared using the same meth-
od as for 13. 15 was obtained in 65.5% yield.
Compound 15: mp: 128–130 ꢁC. Mass spectrum (m/e):
273 (M+). Anal. Calcd for C18H27NO: C, 79.07; H,

K. Shiba et al. / Bioorg. Med. Chem. 14 (2006) 2620–2626
2625
9.95; N, 5.12. Found: C, 78.79; H, 10.11; N, 4.97. Specif-
ic rotation: ½aꢁ ꢀ26.4 (c = 0.568, CHCl3).
5. In vitro competitive binding study
5.1. VAChT binding
30
D
4.10. (+)-2-(4-(4-Methylphenyl)piperidino)cyclohexanol
((+)-p-methylvesamicol) ((+)-PMV) (16)
The assays were performed using a method reported
previously,6 except that 504–710 lg protein of rat
cerebral membranes was used. Briefly, 10 nM
(ꢀ)-[3H]vesamicol was used as a radioligand. Various
concentrations of enantiomers of methylvesamicol ana-
logs or sigma ligands (from 10ꢀ10 to 10ꢀ5 M) were used
as subject compounds. The mixture was incubated for
60 min at 37 ꢁC in the presence of 100 nM haloperidol
to mask sigma receptors.
This compound, 16, was prepared using the same meth-
od as for 14.
Compound 16 was obtained in 30.0% yield.
Compound 16: mp: 128–130 ꢁC. Mass spectrum (m/e):
273 (M+). Anal. Calcd for C18H27NO: C, 79.07; H,
9.95; N, 5.12. Found: C, 78.77; H, 10.02; N, 4.94. Specif-
30
D
ic rotation: ½aꢁ +26.7 (c = 0.500, CHCl3).
5.1.1. r-1 Receptor binding. Rat cerebral membranes
(504–710 lg protein) were incubated in triplicate with
3 nM (+)-[3H]pentazocine and various concentrations
of enantiomers of methylvesamicol analogs or sigma li-
gands (from 10ꢀ10 to 10ꢀ5 M) in 0.5 mL of 50 mM Tris–
HCl (pH 7.8) for 90 min at 37 ꢁC. Nonspecific binding
was determined in the presence of 10 lM (+)-pentazo-
cine. The incubated samples were treated in the same
manner as described for the VAChT binding assays.
4.11. (ꢀ)-2-(4-(2-Trimethylstannylphenyl)piperidi-
no)cyclohexanol ((ꢀ)-o-trimethylstannyl-vesamicol) (19)
To a solution of (ꢀ)-o-iodovesamicol 13 (116 mg
(0.3 mmol)) and hexamethylditin (246 mg (0.75 mmol))
in 5 mL dry toluene was added tetrakis(triphenylphos-
phine)palladium(0) (20 mg (17.5 lmol)). The mixture
was degassed by bubbling argon through the reaction
mixture for 5 min and then refluxed for 15 h under
an argon atmosphere. As the reaction proceeded, the
reaction mixture changed in color from yellow to
black. The black precipitate was concentrated under re-
duced pressure and then purified by preparative thin-
layer chromatography on silica gel using ethylacetate/
hexane (1:5, v/v) as the eluent to yield 19 (42 mg,
33.0%).
5.1.2. r-2 Receptor binding. Rat liver membranes (155–
296 lg protein) were incubated in triplicate with 3 nM
[3H]DTG and various concentrations of enantiomers
of methylvesamicol analogous or sigma ligands (from
10ꢀ10 to 10ꢀ5 M) in 0.5 mL of 50 mM Tris–HCl (pH
7.8) for 90 min at 37 ꢁC in the presence of 1 lM (+)-pen-
tazocine to mask r-1 sites. Nonspecific binding was
determined in the presence of 10 lM DTG and 1 lM
(+)-pentazocine. The incubated samples were treated
in the same manner as described for VAChT binding
assays.
Compound 19, colorless oil. NMR (CDCl3): d 0.30 (9H,
s), 1.42–1.60 (4H, m), 1.60–1.95 (8H, m), 2.02–2.40 (3H,
m), 2.62–3.00 (3H, m), 4.22–4.68 (1H, m), 6.95–7.50
(4H, m). Mass spectrum (m/e): 421,423 [M]+.
6. Data analysis
4.12. (+)-2-(4-(4-Tributylstannylphenyl)piperidino)cyclo-
hexanol ((+)-p-tributylstannyl-vesamicol) (20)
Ki values in inhibition studies and Kd and Bmax values in
saturation binding studies were calculated with the
‘Graphpad Prism’ computer program (GraphPad Soft-
ware, Inc. San Diego, USA).
This compound, 20, was prepared using the same meth-
od as for compound 19 with (+)-p-iodovesamicol 18 and
hexabutylditin. Compound 20 was obtained in 71.2%
yield.
References and notes
Compound 20, colorless oil. NMR (CDCl3): d 0.86–
0.91 (m. 9), 1.00–1.06 (m, 6), 1.12–1.26 (m, 4), 1.29–
1.37 (m, 6), 1.48–1.60 (m, 6), 1.62–1.71 (m, 2),
2.13–2.27 (m, 3), 2.42–2.50 (m, 1), 2.73–2.76 (m, 2),
2.91–2.95 (m, 1), 3.39 (m, 1). Mass spectrum (m/e):
547,549 [M]+.
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